Angiogenesis or neovascularization is the formation of new blood vessels from pre-existing capillaries via a mechanism that involves degradation of the basement membrane which surrounds the parent vessel, migration of endothelial cells through the degraded membrane, proliferation of the migrating cells, endothelial cell differentiation, and loop formation (Folkman, J., Angiogenesis and angiogenesis inhibition: an overview, EXS., 79, 1-8 (1997)). With the exception of wound healing and menstruation, angiogenesis in adults is observed only in pathological situations such as cancer, atherosclerosis, and psoriasis, where it contributes to the progression and symptom manifestation of the disease (Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease, Nat. Med. 1(1), 27-31 (1995)). Other “angiogenesis-related” diseases include endometriosis, Kaposi's sarcoma and other HIV-related conditions, leukemia, scleroderma, pyogenic granuloma, myocardial angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, macular degeneration, and retrolental fibroplasia. As used herein, the term “angiogenesis-related diseases” means pathological conditions that require endothelial cell proliferation for progression and symptom manifestation (Chappey, O., et al. Endothelial cells in culture: an experimental model for the study of vascular dysfunctions. Cell Biol. Toxicol., 12(4-6), 199-205 (1996)).
Increasing experimental evidence suggest that angiogenesis plays an essential role in cancer development. It has been observed that solid tumors neither grow beyond 1-2 mm3 nor metastasize unless they become vascularized (Folkman, J. What is the Evidence that Tumors are Angiogenesis Dependent?, J. Natl. Canc. Inst., 82, 4-6 (1990)). Formation of tumor vasculature is necessary in order to deliver nutrients and oxygen at the tumor site, thus, providing a route for tumor metastasis to distant sites. Compositions that inhibit endothelial cell proliferation and/or migration have been shown to inhibit tumor neovascularization, and to prevent tumor growth and metastasis (Eatock, M. M., et al. Tumour vasculature as a target for anticancer therapy. Cancer Treat Rev. 26(3), 191-204 (2000)). Several of these inhibitors are currently under evaluation in human clinical trials (Deplanque, G., et al. Anti-angiogenic agents: clinical trial design and therapies in development, Eur. J. Cancer, 36, 1713-1724 (2000)).
Antibodies are proteins synthesized by B lymphocytes usually in response to the presence of a foreign substance, called an antigen (Askonas, B. A. Immunoglobulin synthesis and its induction in B-lymphoid cells, Acta Endocrinol Suppl (Copenh), 194, 117-132 (1975)). Antibodies are the recognition elements of the humoral immune response, designed to lyse foreign microorganisms and infected cells via activation of the complement system. Antibodies possess specific affinity for the antigens that induced their formation and they readily complex with them to trigger complement activation. Naturally occurring antibodies consist of two heavy and two light chains linked together by disulfide bonds. Each chain comprises domains of unique sequence responsible for antigen binding (variable domains) and domains of constant sequence involved in complement activation and mediation of antibody-dependent cellular toxicity (constant domains). Furthermore, the variable domains of light (VL) and heavy (VH) chains have similar structure with each domain comprising four somewhat conserved regions, called the framework regions (FR), and three hyper-variable regions, called complementarity determining regions (CDR). Studies have shown that CDRs determine antibody specificity (Ohno et al. Antigen-binding specificities of antibodies are primarily determined by seven residues of VH, Proc Natl Acad Sci USA, 82(9), 2945-2949 (1985)). In VH chains, CDRs are located in the proximity of positions 30-35 (CDR1), 50-65 (CDR2), and 95-102 (CDR3) (Kabat et al. Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no 91-3242 US. Department of Health and Human Services (1991) and Honegger et al.). Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J. Mol. Biol. 309, 657-670 (2001)). In VL chains, CDRs are located in the proximity of positions 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3).
Antibodies produced in response to the presence of a single antigen have a common specificity but they are heterogeneous in nature, since they are derived from many different antibody-producing cells. Homogeneous or monoclonal antibodies can be produced through hybridoma cells (Galfre, G. and Milstein, C. Preparation of monoclonal antibodies: strategies and procedures, Methods Enzymol., 73(Pt B), 3-46 (1981)). The hybridoma cell method of producing large amounts of homogeneous populations of antibodies with a particular specificity has allowed the use of monoclonal antibodies as diagnostic and therapeutic agents (Milstein, C. With the benefit of hindsight, Immunol. Today, 21(8), 359-64 (2000)).
Initially, animal-derived monoclonal antibodies had limited therapeutic value in humans due to antigenicity. The problem was solved with the production of humanized antibodies. Humanized antibodies are defined as immunoglobulin variants or fragments capable of binding to a predetermined antigen and which comprise a FR region having substantially the amino acid sequence of a human immunoglobulin and a CDR region having substantially the amino acid sequence of a non-human immunoglobulin (Hurle, M. R. and Gross, M. Protein engineering techniques for antibody humanization, Curr. Opin. Biotechnol., 5(4), 428-33(1994)). Humanized antibodies have been recently approved for the treatment of various diseases including cancer. Trastuzumab, a humanized antibody against HER-2 receptor, is used for the treatment of breast cancer, while Rituximab, a humanized antibody against CD20, is used for the treatment of lymphoma (Baselga, et al. Phase II study of weekly intravenous trastuzumab (Herceptin) in patients with HER2/neu-overexpressing metastatic breast cancer, Semin. Oncol., 26(4 Suppl 12), 78-83 (1999); Slamon et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2, N. Engl. J. Med., 344(11), 783-92 (2001); Byrd et al. Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity, J. Clin. Oncol., 19(8), 2153-64 (2001)).
Humanized antibodies are usually produced recombinantly. Recombinant production of immunoglobulin variants or fragments requires: a. the isolation and sequencing of the DNA encoding the immunoglobulin variants or fragments and b. the insertion of the isolated DNA into a replicable vector for further cloning (amplification) or expression. DNA encoding fragments of interest is often isolated from cDNA by using appropriate oligonucleotide probes capable of binding to specific genes (e.g., those encoding the heavy and light chains of the monoclonal antibodies). cDNA is obtained by reverse transcription of RNA isolated via conventional methods from hybridoma cells producing the monoclonal antibody of interest. Once isolated, DNA may be placed into a variety of expression vectors, which are then transfected into host cells such as E. coli or Chinese hamster ovary (CHO) cells for intracellular or extracellular production of the antibody variant or fragment of interest. Intracellular production of an antibody or antibody fragment requires its purification from lysates of host cells, while extracellular production requires purification from supernatants of host cells.
Typically, recombinant antibody constructs consist of modified forms of the antigen-binding portion of an antibody, also known as Fv. Single chain Fv molecules (scFv) usually comprise VH and VL domains joined with a small peptide linker in a single polypeptide chain. scFv molecules exhibiting antitumor properties are particularly desired in cancer therapy because their small size (˜30 Kd) allows for tumor penetration. Unfortunately, their small size also facilitates increased blood clearance (Hudson et al. Recombinant antibody constructs in cancer therapy, Curr. Opin. Immunol., 11, 548-557 (1999)). To reduce blood clearance and to increase functional affinity (scFv are monovalent), scFv dimers and trimers have been produced. Formation of multimeric scFv complexes usually depends on the length of the linker between VH and VL domains. Short linkers (5-10 residues) result in the formation of scFv dimers (also known as diabodies), while linkers with less than three residues in length result in the formation of trimers (also known as triabodies) (Holliger et al. “Diabodies”: Small bivalent and bispecific antibody fragments, Proc. Natl. Acad. Sci. USA, 90, 6444-6448 (1993) and Atwell et al. ScFv multimers: length of the linker between VH and VL domain dictates precisely the transition between diabodies and triabodies, Protein Eng., 12, 597-604 (1999)).